The thermophilic esterase from Archaeoglobus fulgidus: Structure and conformational dynamics at high temperature

The esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus is a monomeric protein with a molecular weight of about 35.5 kDa, The enzyme i s barely active at room temperature, displaying the maximal enzyme activity at about 80 degrees C, We have investigated the effect of the temperature on the protein structure by Fourier-transform infrared spectroscopy. The da ta show that between 20 degrees C and 60 degrees C a small but significant decrease of the beta-sheet bands occurred, indicating a partial loss of bet a-sheets, This finding may be surprising for a thermophilic protein and sug gests the presence of a temperature-sensitive beta-sheet, The increase in t emperature from 60 degrees C to 98 degrees C induced a decrease of or-helix and beta-sheet bands which, however, are still easily detected at 98 degre es C indicating that at this temperature some secondary structure elements of the protein remain intact, The conformational dynamics of the esterase w ere investigated by frequency-domain fluorometry and anisotropy decays, The fluorescence studies showed that the intrinsic tryptophanyl fluorescence o f the protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics, Remarkably, the tryptophanyl fluorescence emission reveals that the indolic residues re mained shielded from the solvent up to 80 degrees C, as shown from the emis sion spectra and by acrylamide quenching experiments, The relationship betw een enzyme activity and protein structure is discussed, Proteins 2000;38:35 1-360, (C) 2000 Wiley Liss, Inc.